HIV-I is the most prevalent infectious agent worldwide and a leading causative agent of acquired immuno deficiency syndrome (AIDS). HIV is a retrovirus, which is transmitted through human fluids either through natural interactions like sexual intercourse or through artificial methods like blood transfusions, infected needles used in injections. The 9-10 Kb of HIV-1 genome express viral proteins (categorized in structural, enzymatic and accessory proteins functions), whose function ranges from the successful viral infection in the cell to the complete life cycle for new virions, has been extensively studied. Every viral protein is considered to be a potential target for developing anti-HIV-1 therapy (Greene and Peterlin, 2002; Stevenson 2003).
Further, it was characterized that accessory protein Nef is responsible for HIV-1 pathogenesis (Mellors et. al., 1996) by the experiment where, Nef deleted SIVmac 239 infection in rhesus monkey showed low viral load with normal CD4 counts. These monkeys were found healthy with delayed progression to AIDS (Kestler et. al., 1991). Also, the HIV-1 patients who were long-term non-progressor, with no clinical sign of disease progression and normal CD4 counts, upon characterization of virus revealed that majority of them had Nef gene deleted from their genome (Deacon et. al., 1995, Kirchhoff et. al., 1995,). Interestingly, only expression of Nef gene in mice led to the development of AIDS like phenotype suggesting that it harbors major disease determinant (Hanna et. al., 1998). Nef is deludged with functions involving interaction with different host proteins; however, the individual function required for pathogenesis has not been identified till date.
Nef is a 27 kDa protein, expressed in HIV-1 infected cells even before the formation of provirus suggesting that it initiate functions soon after entry of virus in cell. In vitro and in vivo studies show that Nef protein enhances viral infectivity and replication respectively in infected cells. Nef downregulates the MHC classI from cell surface, which primarily presents viral proteins to activate cytotoxic T lymphocytes. Moreover, Nef functions include activation of Pak kinase, activates cascade of events leading to the activation of T cells without engaging T-cell receptor [TCR] and upregulation of Fas/FasL ligand for killing bystander cells, as well as activates anti-apoptotic pathways for the survival of infected cells. All these functions of Nef are carried out in cell after interaction with host proteins.
The one of the possible mechanism of immune evasion by HIV-1 is increase of expression of both Fas and FasL on virally infected cells (1-3). The expression of FasL is induced by the presence of Nef in virally infected cells (Xu, X. N. et al., 1997. Hodge, S. et al., 1998.). Interestingly, both Fas and FasL presence on infected cells, through cis-ligation, also undergo apoptosis in virally infected cells. The Fas and TNFα-R is known as death receptor signaling pathway which converge to a common signaling molecule known as apoptosis signaling Kinase-1(ASK1). Therefore, ASK1 appears to participate as a key signaling intermediate in both as Fas and TNFα-R pathways, that activated two different subgroup of MAP kinases kinases, SEK1 or (MKK4) and MKK3/MAPKK6(MKK5) which inturn activate JNK; c-Jun amino-terminal kinase and p38 subgroup of MAP kinase, respectively (Chang, H. Y et al., 1998). Ichijo, H. et al., 1997. Nishitoh, H. et al. 1998).
The unique cis-ligated mediated apoptosis in HIV-1 infected cell is inhibited by the presence of Nef. This protein interacts with ASK1 and inhibits the downstream signaling pathways that induce apoptosis. The molecular mechanism studied is Nef interaction with ASK1 appears by specifically preventing stimulus-coupled release of thioredoxin from ASK1 (Geleziunas. R, Xu et al., 2001). possibly, inhibition of thioredoxin release could not activate autophosphorylation of ASK1 kinase by phosphorylating 838 threonine in kinase domain. This cannot activate ASK1 mediated phosphorylation of MEKK-SEK1 kinase which activate JNK pathway (Geleziunas. R, Xu et al., 2001.Hayakawa, T. et al., 2006). The death receptor signaling—ASK1-MEKK-SEK1-JNK axis is involved in activating apoptosis in virally infected cells.
Arguably, if Nef-ASK1 interaction is inhibited then the outcome in the virally infected cells would be falling in the line of apoptosis that helps selectively clearance of HIV-1 infected cell.
Understanding the molecular Nef pathways responsible for functions involved with viral persistence, replication and infectivity can be used as target for anti HIV-1 therapy. This can enable us to focus on future therapies where the task of interrupting key interaction between viral and host proteins will serve as potential target. Nef-ASK1 interaction represents a potential target for this approach.
Recent approaches towards the development of an alternative model for characterizing specific interactions of Nef with host proteins are either yeast (Rossi et. al., 1996) or mammalian-two-hybrid system (Murakami et. al., 2002). Nef is structurally conserved and the structure function analysis revealed that with maintaining the structure, the accessible Nef conserved domain to host proteins is responsible for the function interaction in the cell (Geyer et. al., 2001).
In the present study, we developed a mammalian-two hybrid model and studied Nef-ASK1 interaction in ATCC CRL-1573™ cells, an early event which inhibits death receptor mediated apoptosis in virally infected cells (Geleziunas et al., 2001). Further, the expression of ASK1 fragments showed induction of apoptosis in ATCC TIB-152™/ATCC CRL-1573™. The ASK1 fragment that can induce apoptosis in ATCC TIB-152™/ATCC CRL-1573™ can inhibit apoptosis by the expression of Nef. The molecular mechanism studied showed that the apoptosis was induced by phosphorylation of JNK kinase which was inhibited by the presence of Nef. This model will help to screen the inhibitors designed for this interaction and perhaps, which could be used to develop an alternative therapy.
Mammalian two hybrid models have been used in scientific research in a routine manner (Colas and Brent et al., 1998). The present invention uses this technique to enable the user to establish the Nef-ASK1 interactions in ATCC CRL-1573™ cells. No other group has developed such a model for the evaluation of Nef-ASK1 interactions earlier and the present invention is the first of it's kind. The present invention serves as a novel tool to identify inhibitors of the aforesaid interactions. Inhibitors, which are so identified, will have property to activate apoptosis in cell based system and virally infected cells. There are no known drawbacks to the technique and it is amenable to high-throughput screening. The invention will efficiently allow for the screening of a large number of compounds in a relatively short time period of upto 48 hours. This will also allow for the search of structural analogs of inhibitors identified using the method of the present invention. Inhibitors, which are identified using the present invention, will act through a novel mechanism of inhibiting Nef-ASK1 interaction. This will be an alternate approach for anti-retroviral therapies including but not restricted to the Human immuno deficiency virus (HIV), which causes AIDS.